Ionizing radiation was the first environmental mutagen identified by H.J. Muller in 1928, and radiation is a known human carcinogen. Epidemiological evidence from the Japanese atomic bomb survivors indicates that the female breast is exquisitely sensitive to radiogenic cancer. Women treated for breast cancer are at risk for a new treatment associated breast cancer as are women treated with radiation to the chest for Hodgkin's disease. Recent data indicate that younger women (<age 40) are at a higher risk for radiogenic breast cancer. Our understanding of how radiation acts as a carcinogen is limited. Work by others demonstrated that a transition to genomic instability occurs at the transition from benign hyperplasia to the earliest stages of malignancy in untreated breast biopsy specimens, suggesting that development of an unstable genome is a key step in the etiology of the disease. We will test the central hypothesis that exposure to ionizing radiation (x-rays) will elicit chromosomal instability in the clonal descendants of finite lifespan human mammary epithelial cells (HMEC). This is based on our preliminary finding that irradiation of one strain of finite lifespan HMEC led to karyotypic instability amongst the progeny of single irradiated cells surviving exposure to single doses of 2 Gy or more. The strain of HMEC used (post-selection 184) is from a young donor, and has passed the first senescence barrier with a loss of p16 expression but they retain finite lifespan. Cells of this type are found in focal patches in histologically normal breast tissue, but the majority of finite lifespan HMEC (pre-stasis cells) have not passed the first senescence barrier. Two aims are proposed. Aim 1 tests the hypothesis that ionizing radiation exposure elicits karyotypic instability in post-selection finite lifespan HMEC using cells derived from donors below the age of 40. Aim 2 tests the hypothesis that the establishment of radiation-induced karyotypic instability is reduced in pre-stasis HMEC compared with post-selection finite lifespan HMEC from the same donors evaluated in Aim 1. Mechanistic studies will determine the requirements for silencing of p16INK4a or pRb in the establishment of radiation-induced karyotypic instability. These studies will determine whether radiation-induced karyotypic instability is a rare or a common occurrence after irradiation and whether a transition past the first senescence barrier is required for establishment of the phenotype. PUBLIC HEALTH RELEVANCE: Ionizing radiation is a human carcinogen, and results from the Japanese atomic bomb survivors and medically exposed populations have demonstrated that the young female breast is very sensitive to radiation-induced cancer. Others have shown that genomic instability first appears in human breast biopsy materials at the earliest malignant stage - ductal carcinoma in situ - suggesting that instability is an important early feature in the development of breast cancer. The experiments proposed address whether radiation exposure elicits persistent genomic instability in the progeny of apparently normal finite lifespan human mammary epithelial cells from donors below age 40, whether this is a common phenotype amongst individuals, and whether transition past the first senescence barrier is required for finite lifespan human mammary epithelial cells to exhibit radiation induced genomic instability.